In meso Approaches to Membrane Protein Crystallization - Molecules: Nucleation, Aggregation and Crystallization - page 274

Crystallization from bicelles

Another approach is crystallization from bicelles, which was first applied
to obtain high quality BR crystals (Faham, Bowie, 2002; Faham et al .,
2005). It is known that mixtures of dimyristoylphosphatidylcholine
(DMPC) with certain detergents form lipid-rich bilayer fragments that are
edge-stabilized by the detergent component. The lipid-detergent aggre-
gates in these systems were named bicelles. The most often employed
detergents are either dihexanoylphosphatidylcholine (DHPC) or zwitter-
ionic bile salt derivative, CHAPSO. The 1ipid to detergent ratios present
in the bicellar systems are quite high (usually 4:l to 1.5:l) compared
to other micellar systems which have previously been used as model
membranes in biophysical or biochemical studies (Sanders, Schwonek,
1992; Sanders, Prestegard, 1990). Moreover, the bicellar systems are
unusual in that bicelles can be magnetically oriented to yield oriented
solid state NMR spectra of high quality (Sanders, Landis, 1995).
It is interesting to mention that investigation of bicelle formation is
of great biological importance. It may help us to understand better one
of the fundamental processes in the cell: formation and transformation of
biomembranes.
The procedure of crystallization of membrane proteins from bicelles
can be described as follows:

(i) Prepare 40% (3:1) solution of DMPC/CHAPSO bicelles.

(ii) 10 mg/ml) with the bicelles (v/v ratio of 4:1).

Thereafter, the homogenous mixture contains 8% of the bicelles and
0.8% of the protein. It is believed that at this stage the protein is recon-
stituted into bicelles. These steps are schematically illustrated in Fig. 7.
(iii) In the last step of the preparation of protein crystallization, keep all
solutions on ice (higher temperature increases the viscosity of the
probes). The protein sample is equilibrated against 3.2 M NaH 2 PO 4
solution in a single reservoir by the standard vapor diffusion method.
(iv) Leave the crystallization batch at 37

Mix solubilized BR (C =

° C or room temperature. BR crystals

appear within two weeks.